toxins
Article Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids
Benedict T. Green 1,*, Stephen T. Lee 1, Kevin D. Welch 1, Daniel Cook 1 and William R. Kem 2
1 Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, UT 84341, USA; [email protected] (S.T.L.); [email protected] (K.D.W.); [email protected] (D.C.) 2 Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, 1200 Newell Road, Gainesville, FL 32610-0267, USA; wrkem@ufl.edu * Correspondence: [email protected]; Tel.: +1-435-752-2941
Academic Editor: Peter S. Spencer Received: 29 January 2016; Accepted: 24 June 2016; Published: 4 July 2016
Abstract: Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.
Keywords: anabaseine; anabasine; epibatidine; nicotinic acetylcholine receptor; nicotine; pyridine alkaloid
1. Introduction In grazing livestock, the in utero exposure to certain piperidinyl-pyridine alkaloid plant toxins from Lupinus, Conium, and Nicotiana spp. can cause developmental defects (terata) by inhibiting fetal movement [1]. These defects (arthrogyrposis, kyposis, lordosis, scoliosis, torticollis, and cleft palate) are termed multiple congenital contracture-type (MCC-type) deformities [2]. The mechanism by which these plant toxins inhibit fetal movement has been postulated to involve activation, and then desensitization, of fetal muscle-type nicotinic acetylcholine receptors (nAChRs) that are expressed by the developing fetus [1]. This mechanism is similar to the mechanism of neuromuscular block caused by depolarizing muscle relaxants, like succinylcholine [3]. These alkaloids from Lupinus, Conium, and Nicotiana spp. cause crooked calf syndrome in extensively-grazed cattle in the Western United States. Crooked calf syndrome negatively impacts the welfare of the calf and creates an economic burden due to increased management and veterinary costs.
Toxins 2016, 8, 204; doi:10.3390/toxins8070204 www.mdpi.com/journal/toxins Toxins 2016, 8, 204 2 of 12 Toxins 2016, 8, 204 2 of 12
NicotinicNicotinic acetylcholine acetylcholine receptors receptors are are ligand-gated ligand‐gated cation channels channels and and members members of of the the cys cys-loop‐loop receptorreceptor family. family. Each Each receptor receptor is ais pentameric a pentameric complex complex containing containing five five homologous homologous subunits. subunits. There There are 17are distinct 17 distinct nAChR nAChR subunits; subunits;α1–10 α,1–10β1–4, β,1–4γ,, γδ,, δ and, andε ε [4[4,5],5] that that areare expressed in in vertebrates. vertebrates. More More than than tenten different different subunit subunit combinations combinations have have been been reported reported to occur in in mammals. mammals. In In each each receptor receptor at atleast least twotwo C-loop-containing C‐loop‐containingα α subunitssubunits are are present;present; theythey provide most most of of the the two two acetylcholine acetylcholine binding binding sitessites that that must must be be occupied occupied by by agonists agonists for for maximalmaximal activation.activation. All All nAChRs nAChRs can can exist exist in in multiple multiple conformationconformation states, states, including including resting resting (closed (closed channel),channel), activated (open (open channel), channel), and and desensitized desensitized (agonist(agonist occupied occupied but but closed closed channel) channel) states, states, all of all which of which display display different different allosterically-linked allosterically‐ affinitieslinked affinities for agonists and antagonists [6,7]. Transitions between the various receptor states depend, for agonists and antagonists [6,7]. Transitions between the various receptor states depend, to some to some degree, upon the chemical structure of the agonist. For example, the excitatory degree, upon the chemical structure of the agonist. For example, the excitatory neurotransmitter neurotransmitter acetylcholine (ACh) is more potent at activating fetal muscle‐type nAChR acetylcholine (ACh) is more potent at activating fetal muscle-type nAChR expressed by TE-671 cells expressed by TE‐671 cells than the pyridine alkaloid nicotine, with EC50 values 0.3 and 17.6 μM, than the pyridine alkaloid nicotine, with EC values 0.3 and 17.6 µM, respectively [8]. Prolonged respectively [8]. Prolonged exposure of TE50‐671 cells to ACh also produces a more profound exposuredesensitization of TE-671 than cells nicotine to ACh [9]. also Differences produces in aagonist more profoundactions at desensitizationthe muscle receptor than have nicotine been [ 9]. Differencespartially explained in agonist by actions differences at the in muscle the types receptor of non‐covalent have been bonding partially interactions explained between by differences agonists in theand types the of different non-covalent nAChRs bonding that are interactions related to differences between agonists in the chemical and the structures different nAChRsof the agonists that are related(Figure to differences1A) [10,11]. in the chemical structures of the agonists (Figure1A) [10,11].
Figure 1. Chemical structures of compounds tested in this research (A). Illustration of experimental Figure 1. Chemical structures of compounds tested in this research (A). Illustration of experimental design (B), depicting compound additions one (measuring agonist potency and causing design (B), depicting compound additions one (measuring agonist potency and causing desensitization) desensitization) and two (measuring desensitization), and the KCl addition that served as a and two (measuring desensitization), and the KCl addition that served as a depolarizing calibrant. depolarizing calibrant. The second compound addition was ACh at 10 μM (SH‐SY5Y cells) or 1 μM The second compound addition was ACh at 10 µM (SH-SY5Y cells) or 1 µM (TE-671 cells).The change (TE‐671 cells).The change in relative fluorescence of the dye‐loaded cells was detected using a in relative fluorescence of the dye-loaded cells was detected using a Flexstation III multi-mode Flexstation III multi‐mode microplate reader. microplate reader. In this research, we investigated four nAChR agonists (epibatidine, the most potent known nAChRIn this agonist, research, nicotine, we investigated anabasine, and four anabaseine) nAChR agonists for their (epibatidine, ability to activate the most and potentthen rapidly known nAChRdesensitize agonist, nAChRs. nicotine, The anabasine, rapid type and of desensitization anabaseine) for examined their ability in this to activatestudy has and been then termed rapidly desensitize“classical desensitization” nAChRs. The rapid and develops type of on desensitization a second time scale examined at the agonist in this studyconcentrations has been used termed in “classical desensitization” and develops on a second time scale at the agonist concentrations used in
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Toxins 2016, 8, 204 3 of 12 our investigation [7]. We chose to examine classical desensitization based on our previously reported researchour investigation of plant toxin-induced [7]. We chose inhibition to examine of fetalclassical movement desensitization in goats, based which on has our documented previously reported the nearly immediateresearch abolitionof plant toxin of fetal‐induced goat inhibition movement of by fetal several movement pyridine in goats, alkaloids, which but has not documented nicotine [12 the,13 ]. In thisnearly study immediate we used abolition two cell linesof fetal with goat fetal movement characteristics by several as models pyridine to study alkaloids, receptor but desensitization not nicotine of the[12,13]. two In peripheral this study nAChRs: we used TE-671two cell cells lines expressing with fetal fetalcharacteristics human muscle-type as models to nAChR study (receptorα12β1γδ ) anddesensitization SH-SY5Y cells of derivedthe two peripheral from embryonic nAChRs: central TE‐671 nervous cells expressing system tissuefetal human expressing muscle markers‐type characteristicnAChR (α1 of2β1 immatureγδ) and SH catecholaminergic‐SY5Y cells derived neurons, from embryonic including predominantlycentral nervous autonomic-typesystem tissue nAChRsexpressing containing markersα 3characteristic and β4 subunits of immature [14–17]. catecholaminergic In our experiments, neurons, we used including a dual predominantly agonist addition protocol,autonomic the‐ firsttype agonistnAChRs to containing desensitize α3 and nAChR β4 subunits (see Figure [14–17].1B forIn our a representative experiments, we tracing used a from dual a agonist addition protocol, the first agonist to desensitize nAChR (see Figure 1B for a representative typical experiment depicting initial agonist stimulation), and a second ACh addition to monitor the tracing from a typical experiment depicting initial agonist stimulation), and a second ACh addition degree of desensitization. We report the relative abilities of four pyridine alkaloids (the plant toxins to monitor the degree of desensitization. We report the relative abilities of four pyridine alkaloids nicotine and anabasine, and the animal toxins epibatidine and anabaseine) to activate and rapidly (the plant toxins nicotine and anabasine, and the animal toxins epibatidine and anabaseine) to activate desensitize the two peripheral nervous system nAChRs that have been implicated in MCC-type defect and rapidly desensitize the two peripheral nervous system nAChRs that have been implicated in MCC‐ formationtype defect in fetalformation livestock in fetal and livestock crooked and calf crooked syndrome calf syndrome in grazing in livestock.grazing livestock. 2. Results 2. Results EachEach alkaloid alkaloid acted acted as a as full a nAChRfull nAChR agonist, agonist, as indicated as indicated by the by near the maximal near maximal membrane membrane potential sensingpotential dye sensing fluorescence dye fluorescence for each alkaloid for each in alkaloid the activation-concentration in the activation‐concentration curves forcurves each for cell each line (Figurecell line2, EC (Figure50 values 2, EC presented50 values presented in Table1 ).in Interestingly, Table 1). Interestingly, in the TE-671 in the cells, TE‐671 anabaseine cells, anabaseine was nearly was as potentnearly an as agonist potent as an epibatidine. agonist as epibatidine.
FigureFigure 2. Concentration-response2. Concentration‐response relationships relationships for thefor fourthe four alkaloids alkaloids using using membrane membrane potential potential sensing sensing dye fluorescence in SH‐SY5Y (A) and TE‐671 (B) cells. The cellular responses to agonist were dye fluorescence in SH-SY5Y (A) and TE-671 (B) cells. The cellular responses to agonist were normalized normalized to the maximum epibatidine response for each cell line. The activation concentration‐ to the maximum epibatidine response for each cell line. The activation concentration-response curves response curves are the same curves as in subsequent figures. are the same curves as in subsequent figures. In addition to nAChR activation by the four alkaloids, we also assessed their ability to desensitizeIn addition autonomic to nAChR and activation neuromuscular by the fournAChR alkaloids, subtypes we expressed also assessed by SH their‐SY5Y ability and TE to‐ desensitize671 cells, autonomicrespectively. and neuromuscularThe nAChRs were nAChR classically subtypes desensitized expressed by by exposing SH-SY5Y the and cells TE-671 to five cells, concentrations respectively. Theof nAChRs each alkaloid were classically in log10 molar desensitized increments by exposingfrom 10 nM the cellsto 100 to μ fiveM. concentrationsThe resulting change of each alkaloidin the in logmembrane10 molar potential increments sensing from dye 10 fluorescence nM to 100 µ M.was The measured resulting and change then the in cells the membrane were exposed potential to a
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sensingToxins 2016 dye, 8 fluorescence, 204 was measured and then the cells were exposed to a fixed concentration4 of 12 of ACh (10 µM for SH-SY5Y cells and 1 µM for TE-671 cells) to assess receptor desensitization. All of thefixed four concentration alkaloids were of effectiveACh (10 μ atM desensitizing for SH‐SY5Y the cells responses and 1 μM to for ACh TE‐ in671 both cells) cell to lines assess (Figures receptor3– 6, desensitization. All of the four alkaloids were effective at desensitizing the responses to ACh in both calculated EC50, DC50, and activation/desensitization versus concentration curve intercept values are cell lines (Figures 3–6, calculated EC50, DC50, and activation/desensitization versus concentration presented in Table1). curve intercept values are presented in Table 1).
Table 1. Calculated EC50 and DC50 estimates of compounds in TE-671 and SH-SY5Y cells. Table 1. Calculated EC50 and DC50 estimates of compounds in TE‐671 and SH‐SY5Y cells.
b b ECEC50, 50µ,M SHSH-SY5Y‐SY5Y Cells A/DA/D b ECEC5050 TETE-671‐671 Cells Cells A/DA/D b Compound a a a a Compound a µ a µ µ a µ a µ μM(CI (CI) ) DC50 μM (CI ) InterceptIntercept μM μMM (CI (CI )) DCDC5050 μMM (CI (CI )) InterceptIntercept μMM 0.0180.018 0.015 0.015 0.160.16 0.3 0.3 EpibatidineEpibatidine a 0.020.02 0.30.3 (0.009–0.04) (0.006–0.04) (0.006–0.04) a (0.14–0.2)(0.14–0.2) (0.1–0.6) (0.1–0.6) 3.43.4 1.0 9.59.5 1.9 1.9 NicotineNicotine 0.90.9 44 (1.4–5.1) (0.4–2.7) (0.4–2.7) (7.1–13) (7.1–13) (0.3–12) (0.3–12) 15.315.3 5.6 16.716.7 2.3 2.3 AnabasineAnabasine 88 33 (6.7–67)(6.7–67) (1.7–19) (1.7–19) (13–21) (13–21) (0.7–7.5) (0.7–7.5) 5.95.9 2.4 0.310.31 0.6 0.6 AnabaseineAnabaseine 22 0.40.4 (4.4–36)(4.4–36) (1.0–5.8) (1.0–5.8) (0.2–0.49) (0.2–0.49) (0.2–2.1) (0.2–2.1) aaNinety-five Ninety‐five percent percent confidence confidence interval; interval;b Intercept b Intercept between between the agonist the agonist concentration-effect concentration‐ curveeffect and curve the AChand desensitizationthe ACh desensitization curve. curve.
Figure 3. The actions of epibatidine on the activation and desensitization of nAChR expressed by SH‐ Figure 3. The actions of epibatidine on the activation and desensitization of nAChR expressed by SY5Y (A) and TE‐671 (B) cells. The membrane potential sensing dye fluorescence resulting from the SH-SY5Y (A) and TE-671 (B) cells. The membrane potential sensing dye fluorescence resulting from the addition of an agonist in log10 molar concentrations was measured and displayed as a percentage of addition of an agonist in log10 molar concentrations was measured and displayed as a percentage of the maximal agonist response. Each epibatidine and ACh datum point represents six to 10 the maximal agonist response. Each epibatidine and ACh datum point represents six to 10 experiments experiments of duplicate wells for SH‐SY5Y cells and four experiments of duplicate wells for TE‐671 of duplicate wells for SH-SY5Y cells and four experiments of duplicate wells for TE-671 cells. * p < 0.05, cells. * p < 0.05, epibatidine response versus the cellular response to a fixed concentration of ACh. The epibatidine response versus the cellular response to a fixed concentration of ACh. The vertical dotted vertical dotted lines in each panel are a graphical representation of the A/D intercepts reported in Table 1. lines in each panel are a graphical representation of the A/D intercepts reported in Table1.
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Figure 4. The actions of nicotine on the activation and desensitization of nAChR expressed by SH‐ Figure 4. The actions of nicotine on the activation and desensitization of nAChR expressed by SH-SY5Y SY5Y (A) and TE‐671 (B) cells. The membrane potential sensing dye fluorescence resulting from the A B ( ) and TE-671Figureaddition 4. ( ofThe) an cells. actions agonist The of in membrane nicotine log10 molar on theconcentrations potential activation sensing and was desensitization measured dye fluorescence and ofdisplayed nAChR resulting expressedas a percentage from by SH the of‐ addition of an agonistSY5Ythe maximal in (A log) and10 agonist TEmolar‐671 response.( concentrationsB) cells. TheEach membrane nicotine was measured and potential ACh datumsensing and displayedpoint dye fluorescencerepresents as a percentagefour resulting experiments from of thethe of maximal agonist response.additionduplicate of wells. Each an agonist * p nicotine < 0.05, in nicotinelog and10 molar AChresponse concentrations datum versus point the wascellular represents measured response and four to adisplayed fixed experiments concentration as a percentage of of duplicate ACh. of wells. * p < 0.05,the nicotine maximal response agonist response. versus Each the cellular nicotine and response ACh datum to a fixedpoint concentrationrepresents four experiments of ACh. of duplicate wells. * p < 0.05, nicotine response versus the cellular response to a fixed concentration of ACh.
Figure 5. The actions of anabasine on the activation and desensitization of nAChR expressed by SH-SY5Y (A) and TE-671 (B) cells. The membrane potential sensing dye fluorescence resulting from the
addition of anabasine in log10 molar concentrations was measured and displayed as a percentage of the maximal epibatidine response. Each anabasine and ACh datum point represents four experiments of duplicate wells. * p < 0.05, anabasine response versus the cellular response to a fixed concentration of ACh. Toxins 2016, 8, 204 6 of 12
Figure 5. The actions of anabasine on the activation and desensitization of nAChR expressed by SH‐ SY5Y (A) and TE‐671 (B) cells. The membrane potential sensing dye fluorescence resulting from the
addition of anabasine in log10 molar concentrations was measured and displayed as a percentage of Toxinsthe2016 maximal, 8, 204 epibatidine response. Each anabasine and ACh datum point represents four experiments of6 of 12 duplicate wells. * p < 0.05, anabasine response versus the cellular response to a fixed concentration of ACh.
Figure 6. TheThe actions actions of of anabaseine anabaseine on on the the activation activation and and desensitization desensitization of nAChR of nAChR expressed expressed by SH by‐ SH-SY5YSY5Y (A) (andA) and TE‐671 TE-671 (B) cells. (B) cells. The The membrane membrane potential potential sensing sensing dye dyefluorescence fluorescence resulting resulting from from the addition of anabaseine in log10 molar concentrations was measured and displayed as a percentage of the addition of anabaseine in log10 molar concentrations was measured and displayed as a percentage ofthe the maximal maximal epibatidine epibatidine response. response. Each Each anabaseine anabaseine and and ACh ACh datum datum point point at at each anabaseine concentration represents four experiments of duplicate wells with the exception of the 0.1 μµM ACh datum pointpoint in in TE-671 TE‐671 cells cells which which represents represents three three experiments experiments of duplicate of duplicate wells. * pwells.< 0.05, * anabasinep < 0.05, responseanabasine versus response the versus cellular the response cellular to response a fixed concentration to a fixed concentration of ACh. of ACh.
Epibatidine was the most potent agonist that was tested (Figure 2A,B). It was also the most Epibatidine was the most potent agonist that was tested (Figure2A,B). It was also the most potent potent alkaloid for desensitizing the ACh response (Figure 3A,B). For example, the 10 μM ACh alkaloid for desensitizing the ACh response (Figure3A,B). For example, the 10 µM ACh addition in addition in SH‐SY5Y cells was desensitized at 0.1, 1, 10, and 100 μM (p < 0.05, two‐way ANOVA, SH-SY5Y cells was desensitized at 0.1, 1, 10, and 100 µM(p < 0.05, two-way ANOVA, Holm-Sidak’s Holm‐Sidak’s multiple comparisons test). Similarly, the ACh additions in TE‐671 cells were multiple comparisons test). Similarly, the ACh additions in TE-671 cells were desensitized at 1, 10, and desensitized at 1, 10, and 100 μM (p < 0.05, two‐way ANOVA, Holm‐Sidak’s multiple comparisons 100 µM(p < 0.05, two-way ANOVA, Holm-Sidak’s multiple comparisons test). Moreover, the A/D test). Moreover, the A/D intercept between epibatidine and ACh in both cell lines were the smallest intercept between epibatidine and ACh in both cell lines were the smallest values calculated for any values calculated for any alkaloid reflecting its potency as an agonist (Table 1). alkaloid reflecting its potency as an agonist (Table1). The pyridine alkaloid nicotine does not cause MCC‐type defects because it does not completely The pyridine alkaloid nicotine does not cause MCC-type defects because it does not completely inhibit fetal movement in livestock. It was a potent nAChR agonist in both cell lines (Figure 2A,B). In inhibit fetal movement in livestock. It was a potent nAChR agonist in both cell lines (Figure2A,B). SH‐SY5Y cells which express a neuronal‐type nAChR, the ACh response was desensitized at 10 and In SH-SY5Y cells which express a neuronal-type nAChR, the ACh response was desensitized at 10 and 100 μM by treatment with 10 μM ACh (Figure 4A) (p < 0.05, two‐way ANOVA, Holm‐Sidak’s 100 µM by treatment with 10 µM ACh (Figure4A) ( p < 0.05, two-way ANOVA, Holm-Sidak’s multiple multiple comparisons test). Interestingly, in TE‐671 cells, which express the fetal muscle‐type nAChR, comparisons test). Interestingly, in TE-671 cells, which express the fetal muscle-type nAChR, the the second addition of 1 μM, ACh was also effectively desensitized at 10 and 100 μM concentrations second addition of 1 µM, ACh was also effectively desensitized at 10 and 100 µM concentrations of of nicotine (Figure 4B) (p < 0.05, two‐way ANOVA, Holm‐Sidak’s multiple comparisons test). nicotine (Figure4B) ( p < 0.05, two-way ANOVA, Holm-Sidak’s multiple comparisons test). The piperidinyl-pyridine anabasine, acted as an agonist in both cell lines during its first addition (Figure5A,B). As the concentration of anabasine was increased to 100 µM the response to ACh was desensitized in SH-SY5Y cells (p < 0.05, two-way ANOVA, Holm-Sidak’s multiple comparisons test). Toxins 2016, 8, 204 7 of 12
The piperidinyl‐pyridine anabasine, acted as an agonist in both cell lines during its first addition (FigureToxins 2016 5A,B)., 8, 204 As the concentration of anabasine was increased to 100 μM the response to ACh7 was of 12 desensitized in SH‐SY5Y cells (p < 0.05, two‐way ANOVA, Holm‐Sidak’s multiple comparisons test). In comparison to the neuronal cell line, in the muscle cell line, anabasine was more effective at desensitizingIn comparison the to TE the‐671 neuronal cell response cell line, at 10 in and the 100 muscle μM concentrations cell line, anabasine (p < 0.05, was two more‐way effective ANOVA, at Holmdesensitizing‐Sidak’s multiple the TE-671 comparisons cell response test). at 10 and 100 µM concentrations (p < 0.05, two-way ANOVA, Holm-Sidak’sThe 1,2‐dehydropiperidine multiple comparisons anabaseine test). (Figure 1) was a potent nAChR agonist in both cell lines (FigureThe 6A,B) 1,2-dehydropiperidine but ~20 times more anabaseine potent on the (Figure fetal1 )nAChR was a potent (Table nAChR 1). In SH agonist‐SY5Y incells, both the cell 10 lines μM ACh(Figure response6A,B) but was ~20 desensitized times more potentat 10 and on the 100 fetal μM nAChR anabaseine (Table (1p). < In 0.05, SH-SY5Y two‐way cells, ANOVA, the 10 µ MHolm ACh‐ Sidak’sresponse multiple was desensitized comparisons at 10test). and In 100 TE‐µ671M anabaseinecells the 1.0 ( μp
Figure 7. The response of SH-SY5Y cells to alkaloid agonists in the presence or absence of either 0.1 µM Figure 7. The response of SH‐SY5Y cells to alkaloid agonists in the presence or absence of either 0.1 atropine or 30 nM α-BTx. The membrane potential sensing dye fluorescence resulting from the addition μM atropine or 30 nM α‐BTx. The membrane potential sensing dye fluorescence resulting from the of 0.05 µM epibatidine (A); 10 µM nicotine (B); 10 µM anabasine (C); or 10 µM anabaseine (D) was addition of 0.05 μM epibatidine (A); 10 μM nicotine (B); 10 μM anabasine (C); or 10 μM anabaseine measured and displayed as a percentage of the KCl response. Each column represents the results of experiments with four pairs of duplicate wells in four 96 well plates (n = 4).
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3. Discussion In this research, we found that all of the pyridine alkaloids were full agonists at the autonomic and fetal muscle nAChRs and also desensitized these receptors at concentrations that were usually less than was required for activation. The rank order for activation at the major autonomic (SH-SY5Y cell) nAChR was: epibatidine > nicotine > anabaseine > anabasine. Moreover, the responses of the cells to the agonists was likely due to the activation of autonomic receptors because the responses were insensitive to atropine, which blocks mAChR, or α-BTx, which block α7nAChR (Figure7). This was also the rank order for its rapid desensitization. At the fetal (TE-671 cell) muscle nAChR the rank order for agonist potency was also the same, but there were quantitative differences: while the muscle nAChR potencies of epibatidine and nicotine were reduced and the potency of anabasine was essentially unchanged, the agonist potency of anabaseine was approximately 20 times higher for the fetal nAChR. We included nicotine and epibatidine in this research to serve as mechanistic reference compounds due to the large volume of research that has been done on them. Nicotine and epibatidine interactions with the muscle-type nAChR have been extensively investigated by the Dougherty-Lester Caltech group [11,18]. They have identified intermolecular interactions that result in differences in affinity and potency of cholinergic agonists at muscle-type and certain neuronal nAChRs. The amino acid tryptophan at position 149 (also known as TrpB) of the alpha subunit in the fetal muscle-type nAChR is important for two bonding interactions. First, its tryptophanyl ring forms a cation-π bond with ACh, whereas nicotine does not form a cation-π bond with TrpB at this particular nAChR. Conversely, at certain neuronal nAChRs, both nicotine and ACh form strong cation-π bonds with TrpB in the ligand binding site, and both act as potent agonists [7–11,18,19]. Outside of the central nervous system the lack of cation-π bond interactions at fetal muscle-type nAChRs by nicotine provides a putative explanation for the failure of this alkaloid to inhibit fetal movement and cause MCC-type deformities in livestock [12]. Based on these observations, we speculate that the formation of a cation-π bond with TrpB of the fetal muscle-type receptor expressed by the developing fetus in utero is necessary for potent receptor activation and desensitization, and contributes to the likelihood of MCC-type defects in grazing livestock. These molecular interactions between agonists and the ligand binding sites of the receptors contribute to agonist specific effects at nAChRs. In general, the more potent and effective the initial agonist is at activating a nAChR, the greater, more potent, and effective it is in causing desensitization of the receptor (Table1)[ 7,20]. These results suggest that, based on the TE-671 cell A/D intercept concentrations, anabaseine (0.4 µM) would be more effective than anabasine (3 µM) or nicotine (4 µM) at desensitizing the fetal muscle-type nAChR and, as a result, inhibiting fetal movement through the paralysis of skeletal muscles. In this research we were particularly interested to determine if anabaseine would classically desensitize the ACh response more potently than anabasine. Past research at this laboratory with a mouse bioassay has documented the toxicities of anabaseine and anabasine enantiomers with IV LD50 values of 0.58 ˘ 0.05 mg/kg for anabaseine, 11 ˘ 1 mg/kg R-anabasine, and 16 ˘ 1 mg/kg for S-anabasine, suggesting that, in an acute mouse lethality model, anabaseine is more toxic than anabasine [21]. It is not known what actions anabaseine has on fetal movement in livestock species, such as cattle, sheep, and goats. We have, therefore, used cell-based assays to compare the actions of the known teratogen anabasine to anabaseine. In previous studies with SH-SY5Y, TE-671, and other cells, anabaseine was a more potent agonist than anabasine [22,23]. In the current study, anabaseine was also a more potent agonist than anabasine. As discussed above, the more potent agonists were also more effective at desensitizing nAChRs. The rank order of DC50 values in SH-SY5Y cells, which express autonomic-type nAChR, was epibatidine > nicotine > anabaseine > anabasine. The rank order of DC50 values (Table1) in TE-671 cells, which express fetal muscle-type nAChR, was epibatidine > anabaseine > nicotine > anabasine. The rank order of desensitization was surprising to us because of our previous research with the day-40 pregnant goat model. Results from this work have shown that anabasine abolished all fetal movement at 30 and 60 min after IV dosing of pregnant goats, whereas nicotine only Toxins 2016, 8, 204 9 of 12 slightly reduced fetal movement at 30 and 60 min after IV dosing [12]. Nicotine is well known not to cause MCC-type deformities in livestock (reviewed in [1]). We can only speculate that there may be toxicokinetic differences between anabasine and nicotine in the fetal compartment that may prevent nicotine from inhibiting fetal movement. It seems likely that nicotine is more rapidly metabolized than the other compounds; apparently biotransformation studies of epibatidine, anabasine, and anabaseine have not been reported. However, our results do suggest that anabaseine would be more effective than anabasine at inhibiting fetal movement, based on its lower DC50 value at fetal muscle-type receptors. In conclusion, we show that all four pyridine alkaloids can classically desensitize nAChRs expressed by TE-671 and SH-SY5Y cells. Among the two alkaloids also containing a piperidine ring, anabaseine was more effective than anabasine, which we hypothesize is due to the presence of the imine double bond in the piperidinyl ring of anabaseine. Further research is needed to determine the effectiveness of other piperidine and pyridine alkaloids found in Lupinus, Conium, and Nicotiana spp. at desensitizing peripheral-type nAChRs and to determine how those alkaloids play a role in the formation of fetal defects in livestock species.
4. Conclusions All four pyridine alkaloids were full agonists and classical desensitizers of nAChRs expressed by TE-671 and SH-SY5Y cells. Of the two piperidine-ring containing pyridine alkaloids examined, the 1,2-dehydropiperidine alkaloid anabaseine was much more potent in activating and classically desensitizing nAChRs than anabasine. We hypothesize that the presence of an imine double bond in the piperidinyl ring of anabaseine enhances binding with both types of nAChRs, particularly the muscle type. Further research is needed to determine the effectiveness of other piperidine and pyridine alkaloids found in plants, such as Lupinus, Conium, and Nicotiana spp., at desensitizing nAChR and to determine how those alkaloids play a role in the formation of fetal defects in livestock species.
5. Materials and Methods Epibatidine was purchased from Tocris Bioscience (Bristol, UK); acetylcholine chloride, atropine, and α-BTx were purchased from Sigma-Aldrich (St Louis, MO, USA); and (´)-nicotine ditartrate from EMD Biosciences (La Jolla, CA, USA). Racemic anabasine was isolated from tree tobacco (Nicotiana glauca) and purified in-house by Richard Keeler [22,24]. Anabaseine dihydrochloride was synthesized by Dr. Ferenc Soti (Kem laboratory, University of Florida, Gainesville, FL, USA). Fetal bovine serum was purchased from HyClone Laboratories (Logan, UT, USA); penicillin/streptomycin and Dulbecco’s Modified Eagle’s Medium from Life Technologies (Grand Island, NY, USA); and trypsin-ethylenediaminetetraaacetic acid (EDTA) from American Type Culture Collection (ATCC) (Manassas, VA, USA). Membrane Potential R8034 blue dye kits were obtained from Molecular Devices (Sunnyvale, CA, USA). The SHSY-5Y and TE-671 cell lines were obtained from ATCC (Manassas, VA, USA).
5.1. Cell Culture Cells were grown and maintained in supplemented filter-sterilized growth medium (Dulbecco’s ˝ Modified Eagle’s Medium, 10% fetal bovine serum and 1% penicillin/streptomycin) at 37 C, 5% CO2, in a humidified incubator. Twenty-four hours prior to membrane potential sensing dye fluorescence assays, cells were transferred to 96-well black-walled, clear-bottom assay plates obtained from Corning Incorporated (Corning, NY, USA). Molecular Devices blue dye was dissolved in HBSS-HEPEs (20 mM) at concentration of 1 vial: 360 mL HBSS-HEPES. Both cells and dye were brought to room temperature and the dye loaded into the cell assay plates 30 min prior to measuring changes in fluorescence by a Flexstation III (Molecular Devices Corporation, Sunnyvale, CA, USA) as previously described [22]. Toxins 2016, 8, 204 10 of 12
5.2. Activation and Desensitization Experiments Desensitization is defined as a receptor state that is unresponsive to continued agonist exposure and was first quantitatively described in 1957 by Katz and Thesleff [25]. In this research, a statistically significant decrease (p < 0.05, as denoted by an asterisk in the figures) in the membrane potential sensing dye fluorescence after the second addition of agonist was used as a measure of receptor desensitization. The desensitization assays presented in this research are based upon the work of Campling et al. [26] and Knapman et al. [20]. In our experiments, we used a dual-agonist addition protocol to desensitize nAChR (Figure1B). A baseline fluorescent response was measured for 18 s, then 25 µL of agonist in dye-saline solution was added, and the membrane potential sensing dye fluorescence measured. The membrane potential sensing dye fluorescence was then allowed to stabilize before a second 25 µL addition of agonist; in this case, a maximally stimulating ACh containing saline at 98 s (10 µM ACh for SH-SY5Y cells and 1 µM ACh for TE-671 cells). The ACh concentration was based on concentration-effect experiments that identified the lowest concentration of ACh that provided a maximum membrane potential sensing dye fluorescence in each cell line (Figure S1). At 180 s into the experiment, 25 µL of KCl, in dye-saline solution, was added to attain a final concentration of 40 mM KCl for complete depolarization of the cells. Fluorescent readings were taken every 1.12 s for 255 s with a total of 228 readings per well using a Flexstation III with wavelength excitation at 530 nm, emission of 565 nm, and a cut off wavelength of 550 nm. Compound responses were calculated as previously described [21]. For experiments with atropine and α-BTx, the SH-SY5Y cells were prepared as above and then manually exposed to the antagonists just prior to placing them onto the Flexstation for agonist addition and fluorescence detection.
5.3. Statistical Analysis The membrane potential sensing dye fluorescence of the cells was normalized to the maximum response generated by each agonist in each concentration effect curve with Prism version 6.03 (GraphPad Software, San Diego, CA, USA, 2015), except for Figure2, where the responses were normalized to epibatidine, as previously described [8] to allow for a visual comparison of alkaloid agonism. Fifty percent effective concentration (EC50) and fifty percent desensitization (DC50) values were calculated using nonlinear regression analysis in Prism, as previously described, using the log (agonist) versus a normalized response-variable slope equation. To calculate the agonist concentration of the mid-point of the “smoldering activation” concentration range [26], the percent response of the A/D intercept was estimated by drawing a line from the intercept to the y-axis and rounding the response to the nearest whole percent. That percent response value, the EC50 concentration, and the hill slope were used with the ECanything calculator from GraphPad software [27] to estimate the A/D intercept presented in Table1. A two-way ANOVA was used to detect differences between multiple means using a Holm-Sidak’s multiple comparisons test which was corrected for multiple comparisons. A one-way ANOVA was used to analyze cell response to the presence or absence of antagonists. In all cases, the limit for statistical significance was set at p < 0.05.
Supplementary Materials: The following are available online at www.mdpi.com/2072-6651/8/7/204/s1, Figure S1: The concentration-response relationships for ACh using membrane potential sensing dye fluorescence in SH-SY5Y (A) and TE-671 (B) cells. The cellular responses to agonist were normalized to the maximum epibatidine response for each cell line. Acknowledgments: The authors thank Jim Pfister for his statistical advice, Isabelle McCollum, for her helpful comments and technical expertise, and David R. Brown for his advice in preparing the manuscript. This research was supported by United States Department of Agriculture, Agricultural Research Service (USDA/ARS). Author Contributions: Benedict T. Green and William R. Kem conceived of the study, analyzed and interpreted the data. Stephen T. Lee provided the anabasine. William R. Kem provided the anabaseine. Kevin D. Welch and Daniel Cook conceived of experiments, and provided data analysis and interpretation. All authors participated in the writing and revising of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Toxins 2016, 8, 204 11 of 12
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